Detection of hydroxymethylcytosine bases

ABSTRACT

Methodologies for labeling the epigenetic modification 5-hydroxymethyl-cytosine (5hmC) along a DNA molecule, and for determining a presence or a level of this epigenetic modification based on a ratio of fluorescence intensity of a labeled DNA sample to absorption intensity of the DNA sample at 260 nm are disclosed. Related compositions and reagents, and methods of preparing same are also disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/424,523, filed on May 29, 2019, which is a division of U.S. patentapplication Ser. No. 14/894,350 filed on Nov. 26, 2015, now U.S. Pat.No. 10,351,898, which is National Phase of PCT Patent Application No.PCT/IL2014/050010 having International Filing Date of Jan. 5, 2014,which claims the benefit of priority under 35 USC § 119(e) of U.S.Provisional Patent Application No. 61/828,129 filed on May 28, 2013. Thecontents of the above applications are all incorporated by reference asif fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 88954SequenceListing.txt, created on Aug. 12,2021, comprising 1,431 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to molecularbiology and, more particularly, but not exclusively, to detection andmapping of 5-hydroxymethyl cytosine bases within single or plurality ofDNA molecules.

Epigenetics refers to DNA and chromatic modifications that persist fromone cell division to the next without change in the underlying DNAsequence. These dynamic, chemical modifications are a major source ofgenomic variation, yet this variation is difficult to detect by currenttechnologies since it is masked by ensemble averaging.

Epigenetic modifications include cytosine methylation (5mC) and therecently discovered cytosine hydroxymethylation (5hmC), which exhibitstissue and cell type specific distribution in mammalian genomes.

In recent studies of genomic DNA from human and mouse brain tissue andmouse embryonic stem cells, it was found that a substantial fraction of5-methyl-cytosine (5mC) in CpG dinucleotides is converted to 5hmC by theaction of the Tet family Fe(II)-dependent oxygenases. The distributionof 5hmC in mammals is tissue specific and non-random, suggesting thatits deposition is highly regulated and that it may have a functionalrole in transcription regulation. Today, 5hmC is widely accepted as thesixth base of DNA (after 5-methylcytosine, the fifth base), and it is inthe focus of extensive research.

To elucidate the role of 5hmC, information regarding quantity anddistribution is critical, and several methods for the specific detectionof 5hmC have been reported since its discovery in mammalian tissue in2009 [M. Münzel, D. Globisch, and T. Carell, Angewandte Chemie(International ed. in English), 2011, 50, 6460-8].

Selective functionalization of 5hmC is based on the discovery that T4β-glucosyltransferase (β-GT) from T-4 bacteriophages can attach aglucose moiety from uridine diphosphoglucose (UDP-Glu) onto the hydroxylgroup of 5hmC, resulting in a glucosylated nucleotide. Song et al. [inNature biotechnology, 2011, 29, 68-72 and U.S. patent application havingPublication No. 2011/0301045] utilized this enzymatic process totransfer a glucose chemically modified with an azide group onto 5hmC ingenomic DNA. Using Huisgen cycloaddition (click) chemistry, theyattached a biotin to the azide group and captured the 5hmC-containingDNA on streptavidin-coated magnetic beads for sequencing. A commerciallyavailable product, the Hydroxymethyl Collector™ (by Active Motif), fordetecting and capturing DNA fragments containing 5-hmC methylation, wasdeveloped based on this methodology.

However, due to the short sequence reads, sequencing reports on thepopulation averaged distribution of 5hmC cannot resolve smallsub-populations or characterize variation in the 5hmC patterns, whichare required for identifying epigenetic modifications which may displayhigh cell to cell variation due to their dynamic nature.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of labeling the epigenetic modification5-hydroxymethyl-cytosine (5hmC) along a DNA molecule:

(a) attaching to the DNA molecule a 5hmc specific labeling agent; and

(b) extending the DNA molecule.

According to some embodiments of the invention, the extending islinearly extending.

According to some embodiments of the invention, step (b) is effectedfollowing step (a).

According to some embodiments of the invention, the method furthercomprises attaching to the DNA molecule an additional labeling agentdistinct of the 5hmc specific labeling agent.

According to some embodiments of the invention, the additional labelingagent is a 5mc specific labeling agent.

According to some embodiments of the invention, the additional labelingagent is an epigenetic modification specific labeling agent.

According to some embodiments of the invention, the additional labelingagent is a non-epigenetic modification specific labeling agent.

According to some embodiments of the invention, the does not comprisesubjecting the DNA molecule to fragmentation.

According to some embodiments of the invention, the extending iseffected by depositing the DNA molecule on a surface or extending theDNA molecule in a nanochannel.

According to some embodiments of the invention, the method furthercomprises identifying a position of the 5-hydroxymethyl-cytosine (5hmC)along the DNA molecule.

According to some embodiments of the invention, attaching the labelingagent comprises:

reacting a labeling agent derivatized by a second reactive group with aDNA molecule in which the 5-hydroxymethylcytosines are glycosylated by aglucose molecule derivatized by a first reactive group, wherein thefirst and second reactive groups are chemically compatible to oneanother.

According to some embodiments of the invention, glycosylating the5-hydroxymethylcytosines in the DNA molecule comprises incubating theDNA molecule with β-glucosyltransferase and a uridine diphosphoglucose(UDP-Glu) derivatized by the first reactive group.

According to some embodiments of the invention, one of the first andsecond reactive groups is azide and the other is alkyne, such thatattaching the labeling agent to the DNA molecule is effected by a clickchemistry.

According to some embodiments of the invention, the reacting is free ofa copper catalyst.

According to some embodiments of the invention, the first reactive groupis azide.

According to some embodiments of the invention, the uridinediphosphoglucose (UDP-Glu) derivatized by the first reactive group is aUDP-6-N₃-Glucose. According to some embodiments of the invention, theUDP-6-N₃-Glucose is synthesized chemically.

According to some embodiments of the invention, the UDP-6-N₃-Glucose issynthesized enzymatically.

According to some embodiments of the invention, the labeling agent is afluorescent labeling agent.

According to an aspect of some embodiments of the present inventionthere is provided a method of in-situ imaging a DNA molecule, the methodcomprising:

(a) attaching a labeling agent to the DNA molecule according to themethod of any one of claims 11-15; and

(b) subjecting the DNA molecule to an imaging method suitable fordetecting the labeling agent.

According to some embodiments of the invention, the labeling agent is afluorescent agent and the imaging method is a fluorescence imaging.

According to some embodiments of the invention, the method furthercomprises generating an optical image of the DNA molecule following theimaging.

According to an aspect of some embodiments of the present inventionthere is provided an extended DNA molecule comprising at least one5hmc-specific labeling agent.

According to an aspect of some embodiments of the present inventionthere is provided a DNA molecule comprising at least two differentlabeling agents, wherein a first labeling agent of the at least twodifferent labels is a 5hmc-specific labeling agent.

According to some embodiments of the invention, the 5hmc-specificlabeling agent is attached to the DNA molecule by reacting a labelingagent derivatized by a second reactive group with a DNA molecule inwhich the 5-hydroxymethylcytosines are glycosylated by a glucosemolecule derivatized by a first reactive group, wherein the first andsecond reactive groups are chemically compatible to one another.

According to some embodiments of the invention, one the first and secondreactive groups is azide and the other is alkyne, such that attachingthe labeling agent to the DNA molecule is effected by a click chemistry.

According to some embodiments of the invention, the reacting is free ofa copper catalyst.

According to some embodiments of the invention, the first reactive groupis azide.

According to some embodiments of the invention, the labeling agent is afluorescent labeling agent.

According to some embodiments of the invention, a second labeling agentof the at least two different labeling agents is a 5mc-specific labelingagent.

According to some embodiments of the invention, a second labeling agentof the at least two different labeling agents is for an epigeneticmodification.

According to some embodiments of the invention, a second labeling agentof the at least two different labeling agents is for anon-epigenetically modified base.

According to some embodiments of the invention, the DNA molecule isextended.

According to some embodiments of the invention, the DNA molecule is agenomic DNA molecule.

According to some embodiments of the invention, the DNA molecule islonger than 20 Kb.

According to some embodiments of the invention, the DNA molecule islonger than 30 Kb.

According to some embodiments of the invention, the DNA molecule islonger than 40 Kb.

According to an aspect of some embodiments of the present inventionthere is provided a method of detecting 5-hydroxymethyl-cytosine (5hmC)in a DNA sample the method comprising:

(a) reacting the DNA sample with a 5hmc-specific fluorescent agent underconditions which allow staining of the DNA sample with the 5hmc-specificlabeling agent so as to obtain a 5hmC-labeled DNA sample; and

(b) measuring fluorescence intensity of the 5hmC-labeled DNA sample (X)and absorption intensity of the DNA, at 260 nm (Y), wherein a ratiobetween X to Y is indicative of presence or level of 5hmC in the DNAsample.

According to some embodiments of the invention, the ratio is compared toa ratiometric calibration curve.

According to some embodiments of the invention, the detecting iseffected in a high throughput setting of at least 300 DNA samples.

According to an aspect of some embodiments of the present inventionthere is provided a composition-of-matter comprising the DNA molecule,as described herein.

According to some embodiments of the invention, the DNA molecule issurface deposited or extended in a microchannel.

According to an aspect of some embodiments of the present inventionthere is provided a method of preparing UDP-6-N₃-Glucose, the methodcomprising subjecting an azido glucose (6-azido glucose) to an enzymaticcatalysis by kinase N-acetylhexoseamine 1-kinase (NahK), in the presenceof ATP to thereby obtain a phosphorylated 6-azidoglucose; and subjectingthe phosphorylated 6-azido glucose to enzymatic catalysis byuridyltransferase (GlmU) in the presence of UTP.

According to an aspect of some embodiments of the present inventionthere is provided a computer readable storage medium comprising adatabase including a plurality of DNA sequences and informationpertaining to 5hmC modification of the plurality of DNA sequences.

According to some embodiments of the invention, wherein the informationis selected from the group consisting of, position of the 5hmC, level ofthe 5hmC, tissue distribution of the 5hmC.

In view of the limitations in detecting small sub-populations of 5hmCmodifications and/or in characterizing variation in the 5hmC patterns,which are associated with variations in the distribution of 5hmC, a needfor a single-molecule detection of 5hmC-modified DNA has beenrecognized.

The present inventors have designed and successfully practiced amethodology for specific labelling of the epigenetic modification5-hydroxymethyl-cytosine along genomic DNA molecules with a labelingagent such as a fluorescent reporter molecule. The disclosed methodologyis based on enzymatic glucosylation followed by a click chemistryreaction, and enables single molecule detection as well as globalquantification of 5hmC in genomic DNA.

Embodiments of the invention relate to methods of labeling DNA andimaging the labeled DNA molecule at the single molecule level whilemaintaining high sensitivity. The ability to specifically labelepigenetic information holds promise in single-molecule DNA barcodingapplications. These methods bridge the gap between the single-baseresolution but short reads of sequencing methods and the long rangegenomic context but low resolution (Mbp) of cytogenetic techniques suchas chromosome fluorescence in situ hybridization (FISH). An emergingtechnology that utilizes DNA barcoding is optical mapping. Thistechnique uses fluorescence imaging of linearly extended DNA moleculesto probe information patterns along the molecules. Before imaging, DNAis deposited on surfaces or extended in nanochannels, andsequence-specific information, such as locations of enzymaticrecognition sites, is readout along the DNA like beads on a string. Thisbarcoding technique is focused on sequence-specific labeling thatprovides information regarding the genetic identity of the observedmolecule. The detected pattern can be used as a scaffold for assemblingand finishing sequencing data but also to detect structural variations.The ability to simultaneously record epigenetic information such as theDNA modifications 5mC and 5hmC as well as the distribution of histonesand transcription factors, may reveal long-range epigenetic patternsalong individual chromosomes and highlight genomic variation hidden orinaccessible by traditional techniques.

Using the methodology disclosed herein, a simple and quick UV-vismeasurement replaces currently used radioactive, mass-spec and affinitybased methods.

The methodology described herein can be used as a substitute toimmunostaining in histological samples, tissue sections, chromosomes andother cytogenetic applications.

Single molecule optical patterns may serve as biomarkers for earlydiagnostics as well as for monitoring the bioactivity of drugs thatinfluence hydroxymethylation.

Embodiments of the present invention further relate to a preparation ofUDP-azide glucose, an exemplary reagent useful in the methodologydescribed herein, for specifically labeling 5hmC in a DNA molecule, viasimple enzymatic reactions.

The UDP-azide glucose can be used for introducing an azide (reactive)group to hydroxymethylcytosine to which various functional groups can beattached via click chemistry. Such functional groups include biotin orother affinity tags as well as various contrast agents such asradiolabeling agents and isotopic labeling agents.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a scheme showing an exemplary synthesis of a 6-azido glucose,according to some embodiments of the present invention.

FIG. 2 is a scheme showing 5hmC labeling by enzymatic glucosylation,followed by a click chemistry reaction to thereby attach selectively alabeling agent (e.g., ALEXA FLUOR®) to 5hmc, according to exemplaryembodiments of the present invention.

FIGS. 3A-3D are images showing single molecule detection of 5hmC. FIGS.3A-3B: Mapping of 5hmC sites on lambda phage genomes. FIG. 3A is ahistogram built from the 5hmC position maps of 93 genomes. A combinationof eight Gaussians (red dashed line) centered on the expected 5hmC sites(black solid line) nicely represents the experimental data. FIG. 3B is asingle-molecule image of lambda phage genomes (green) labeled with ALEXAFLUOR®) 555 (red). The optical patterns match the expected optical map(red dots, upper panel). FIGS. 3C-3D are single molecule images of DNAextracted from mouse tissues. DNA (green) was extracted from brain (FIG.3C) and kidney (FIG. 3D) and 5hmC was labeled with Cy5 (red). (scale bar10 μm/˜35 Kbp).

FIGS. 4A-4B are graphs showing quantification of 5hmC levels in solubleDNA samples. FIG. 4A shows an absorbance spectrum of a PCR-amplified,70-bp DNA molecule containing three 5hmC residues, labeled with ALEXAFLUOR®) 647. The absorption peaks at 260 nm and 650 nm correspond to theabsorption maxima of DNA and of ALEXA FLUOR®) 647 respectively. FIG. 4Bis a calibration curve of the ratio between the absorbance at 260 nm(nucleotide bases) and at 650 nm (5hmC-ALEXA FLUOR® 647 label) as afunction of % 5hmC per total nucleotides. Each data point represents anaverage of three measurements.

FIGS. 5A-5B are graphs showing absorption spectrum of DNA (FIG. 5A), andfluorescence emission spectrum (FIG. 5B) of hmC labeled with Cy5 by theclick reaction from different tissues.

FIG. 6A is a graphic presentation showing the percent ofhydroxymethylated cytosine out of total nucleotides in various tissues.Samples were scanned from a 384 well plate, by a plate reader. The % hmCwas calculated from the ratio of the fluorescence at 670 nm and theabsorption at 260 nm, which is compared to a calibration cure (insert)prepared with known % hmC. Red dots represents a calibration curveprepared from DNA with known percentage of hmC, labeled with Cy5following the enzymatic and click reaction. The blue dots represent acalibration curve made of DNA fragments containing known percentage ofCy5. These fragments are made by PCR with Cy5-labeled cytosines, amongthe other nucleotides. The ratio between the slope of these twocalibration curves indicate the efficiency of the hmC labeling reaction.

FIG. 6B is a heat map of the ratio F670/A260 for the given samples as itis scanned by the plate reader.

FIGS. 7A-7C are images showing single DNA fragments (green stringslabeled with YoYo-1) stretched on modified glass slides and labeled forhmC with Cy5 (red dots). DNA was extracted from PBMC (FIG. 7A), spleen(FIG. 7B) and brain (FIG. 7C) tissues. Blue arrows point to fragmentscontaining high hmC density and Orange arrows for fragments containinglow hmC labels.

FIGS. 8A-8B are images showing DNA fragments stretched on a glass slide,extracted from Zebra fish (FIG. 8A) and from mouse brain (FIG. 8B),labeled with YoYo-1 (blue). Click chemistry was used for the labeling ofhmC sites with Cy5 (pink) and the nicked regions, forming the DNAbarcoding are labeled with Atto 550 (green).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to molecularbiology and, more particularly, but not exclusively, to detection andmapping of 5-hydroxymethyl cytosine bases within a DNA molecule.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The epigenetic modification 5-hydroxymethyl-cytosine is a DNA pyrimidinenitrogen base. A methyl group and then a hydroxy group are added to acytosine base. It is important in epigenetics, because the hydroxymethylgroup on the cytosine can possibly switch gene expression. To date theexact role of 5hmC is poorly understood. Thus, elucidating the role of5hmC and gathering information regarding the quantity and distributionof this nucleotide is critical.

Whilst conceiving the present invention, and reducing it to practice,the present inventors have devised a specifically detectable agent for5hmC which can be used to label 5hmC along genomic DNA molecules with afluorescent reporter molecule. Enzymatic glucosylation followed by aclick chemistry reaction enables single molecule detection as well asglobal quantification of 5hmC in genomic DNA.

As is illustrated hereinbelow and in the Examples section which follows,the present inventors have devised a novel method for labeling anddetecting 5hmC sites with a fluorescent reporter molecule (labelingagent) in which DNA molecules can be imaged at the single molecule leveland a plurality of DNA molecules can be analyzed in high throughputsettings. An exemplary labeling scheme is provided in FIG. 2 and inExample 1 below. As shown, a glucosyltransferase is fed with a syntheticcofactor UDP-6-N₃-Glu, resulting in covalent attachment of a functionalazide at the 5hmC site (FIG. 2).

This azide is further reacted with an ALEXA FLUOR®) alkyne via a “click”chemistry reaction to generate the fluorescently labeled 5hmC (FIG. 2).The resulting DNA product has fluorescence and absorbance proportionalto the content of 5hmC residues.

The present inventors have exploited the ability to specifically labelepigenetic information in single-molecule DNA barcoding applications.These methods bridge the gap between the single-base resolution butshort reads of sequencing methods and the long range genomic context butlow resolution (Mbp) of cytogenetic techniques such as chromosomefluorescence in situ hybridization (FISH).

Thus, the present inventors have further used optical imaging in orderto detect 5hmC at the individual molecule level. This technique employsfluorescence imaging of linearly extended DNA molecules to probeinformation patterns along the molecules. Before imaging, labeled DNA isdeposited on surfaces or extended in nanochannels, and sequence-specificinformation, such as locations of 5hmC, enzymatic recognition sites,histones and the like is readout along the DNA like beads on a string(see Example 1). The use of fluorescent dyes that aresequence-specifically incorporated such as by an enzymatic reaction andthat are imaged as fluorescent spots along the DNA result in barcodingtechniques which provide information regarding the genetic identity ofthe observed molecule. The ability to simultaneously record epigeneticinformation such as the DNA modifications 5mC and 5hmC as well as thedistribution of histones and transcription factors, may reveallong-range epigenetic patterns along individual chromosomes andhighlight genomic variation hidden or inaccessible by traditionaltechniques.

Using the present teachings, the present inventors were able to detect asingle label within a 50-kb genome corresponding to a 5hmC content of0.002%, demonstrating unprecedented sensitivity.

While further reducing the present invention to practice, the presentinventors have realized that calculating the fluorescence intensity(rather than the absorption) of a hmC-labeled DNA sample and theabsorption intensity of the DNA, at 260 nm (FIGS. 5A-5B) provides verysensitive detection which is far higher than that detected by absorptionmeasurements of labeled 5hmC, allowing to detect down to 0.004% hmC/dNfrom a DNA sample such as extracted from liver. This method negates theneed for DNA immobilization thus rendering it technically accessible,cost effective and especially useful for determining 5hmC levels in highthroughput settings.

In summary, a simple, fast, and cost-effective method is presented forthe specific labeling of 5hmC with fluorescent reporter molecules. Thismethod can be used to label engineered as well as native 5hmC sites ongenomic DNA and demonstrate the potential of fluorescent labeling forsingle-molecule mapping of 5hmC patterns along genomic DNA. In addition,the labeling allows rapid screening and quantification of global 5hmClevels in genomic DNA using conventional UV-vis spectrophotometry withsensitivity that rivals currently existing methods for global 5hmCquantification.

Thus, according to an aspect of some embodiments of the presentinvention there is provided a method of labeling the epigeneticmodification 5-hydroxymethyl-cytosine (5hmC) along a DNA molecule.

As used herein “5-Methylcytosine” or “5mC” is a methylated form of theDNA base cytosine. When cytosine is methylated, the DNA maintains thesame sequence, but the expression of methylated genes can be altered(the study of this is part of the field of epigenetics).5-Methylcytosine is incorporated in the nucleoside 5-methylcytidine.

As used herein “5-Hydroxymethylcytosine” or “5hmC” is a DNA pyrimidinenitrogen base. It is formed from the DNA base cytosine by adding amethyl group and then a hydroxy group.

As used herein the term “DNA” refers to single stranded DNA or a doublestranded DNA which is isolated. The DNA can be a eukaryotic DNA (e.g.,rodent or primate e.g., human) in which 5hmC modifications typicallyoccur or a synthetic DNA in which 5hmC modifications may be artificiallyadded.

According to an embodiment of the invention, the DNA molecule is acomplementary polynucleotide sequence (cDNA) to which 5hmC modificationshave been artificially added, a genomic polynucleotide sequence and/or acomposite polynucleotide sequences (e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the polypeptide of the present invention,as well as some intronic sequences interposing therebetween. Theintronic sequences can be of any source, including of other genes, andtypically will include conserved splicing signal sequences. Suchintronic sequences may further include cis acting expression regulatoryelements.

The length of the DNA molecule may vary. Exemplary ranges include, butare not limited to 1-15,000 Kb, reflecting at the high range the size ofa human chromosomes (or chromatin).

According to some embodiments of the invention, the DNA molecule islonger than 20 Kb.

According to some embodiments of the invention, the DNA molecule islonger than 30 Kb.

According to some embodiments of the invention, the DNA molecule islonger than 40 Kb.

Detection of the labeled DNA molecule can be done at the single moleculelevel (see FIGS. 3A-3D, 7A-7C and 8A-8B) using optical imaging asfurther described hereinbelow. Alternatively, detection of labeled DNAmolecules can be done at the global level, analyzing the presence orlevel of 5hmC modification of a plurality of DNA molecules at the cell,tissue and organism level (as shown in FIGS. 5A-5B), as furtherdescribed hereinbelow.

Thus, according to an embodiment of the invention there is provided amethod of labeling the epigenetic modification 5-hydroxymethyl-cytosine(5hmC) along a (single) DNA molecule, the method comprising:

(a) attaching to the DNA molecule a 5hmc specific labeling agent; and(b) extending the DNA molecule.

As mentioned hereinabove and further described hereinbelow, attachmentof a 5hmc specific labeling agent to the DNA molecule is effected whenanalysis is performed in the single molecule level or when a pluralityof DNA molecules (global 5hmC analysis) are analyzed.

As used herein “a 5hmC specific labeling agent” refers to a labelingagent that differentiates between 5hmC modification and non-modifiedcytosine or methylated cytosine (5mC), as described hereinabove. A 5hmCspecific labeling agent labels selectively the position or positionswhere 5hmC modification is present in a DNA molecule, and does not labelthose positions in a DNA molecule where 5mC or any other epigeneticmodification is present. The 5hmC labeling agent according to someembodiments of the present invention is fluorescently detectable. A listof suitable labeling agents is provided hereinafter.

According to some embodiments of the invention, a 5hmC specific labelingagent labels at least 50%, or at least 70%, or at least 80%, or at least90% of the a 5hmC modifications in a DNA molecule, including anyintermediate within 50-100%.

According to some embodiments of the present invention, a 5hmC specificlabeling agent is attached (e.g., covalently) selectively to 5hmC.

In some embodiments, selectively attaching a 5hmC specific labelingagent is effected by:

reacting a labeling agent derivatized by a reactive group (hereinreferred to as a second reactive group) with a DNA molecule in which the5-hydroxymethylcytosine bases are glycosylated by a glucose moleculederivatized by another reactive group (herein referred to as a firstreactive group).

The first and second reactive groups are selected as being chemicalcompatible to one another.

By “chemically compatible” it is meant that the first and secondreactive groups can react with one another so as to form a chemicalbond.

As used herein, the phrase “reactive group” describes a chemical groupthat is capable of undergoing a chemical reaction that typically leadsto a bond formation. The bond can involve one or more of a covalentbond, an electrostatic bond, a hydrogen bond, aromatic interactions, andany combination thereof.

The bond, according to some embodiments of the present invention, is acovalent bond.

Chemical reactions that lead to a bond formation include, for example,cycloaddition reactions (such as the Diels-Alder's reaction, the1,3-dipolar cycloaddition Huisgen reaction, and the similar “clickreaction”), condensations, nucleophilic and electrophilic additionreactions, nucleophilic and electrophilic substitutions, addition andelimination reactions, alkylation reactions, rearrangement reactions andany other known organic reactions that involve a reactive group.

Representative examples of reactive groups include, without limitation,acyl halide, aldehyde, alkoxy, alkyne, amide, amine, aryloxy, azide,aziridine, azo, carbamate, carbonyl, carboxyl, carboxylate, cyano,diene, dienophile, epoxy, guanidine, guanyl, halide, hydrazide,hydrazine, hydroxy, hydroxylamine, imino, isocyanate, nitro, phosphate,phosphonate, sulfinyl, sulfonamide, sulfonate, thioalkoxy, thioaryloxy,thiocarbamate, thiocarbonyl, thiohydroxy, thiourea and urea, as theseterms are defined hereinafter.

Exemplary first and second reactive groups that are chemicallycompatible with one another as described herein include, but are notlimited to, hydroxy and carboxylic acid, which form an ester bond; thioland carboxylic acid, which form a thioester bond; amine and carboxylicacid, which form an amide bond; aldehyde and amine, hydrazine,hydrazide, hydroxylamine, phenylhydrazine, semicarbazide orthiosemicarbazide, which form a Schiff base (imine bond); alkene anddiene, which react therebetween via cycloaddition reactions; andreactive groups that can participate in a Click reaction.

Additional examples of pairs of reactive groups (first and secondreactive groups) capable of reacting with one another include an azideand an alkyne, an unsaturated carbon-carbon bond (e.g., acrylate,methacrylate, maleimide) and a thiol, an unsaturated carbon-carbon bondand an amine, a carboxylic acid and an amine, a hydroxyl and anisocyanate, a carboxylic acid and an isocyanate, an amine and anisocyanate, a thiol and an isocyanate. Additional examples include anamine, a hydroxyl, a thiol or a carboxylic acid along with anucleophilic leaving group (e.g., hydroxysuccinimide, a halogen).

It is to be appreciated that for each pair of reactive groups describedhereinabove, either reactive group can correspond to the “first reactivegroup” or to the “second reactive group”.

In some embodiments, the first and/or the second reactive groups can belatent groups, which are exposed during the chemical reaction, such thatthe reacting (e.g., covalent bond formation) is effected once a latentgroup is exposed. Exemplary such groups include, but are not limited to,reactive groups as described hereinabove, which are protected with aprotecting group that is labile under selected reaction conditions.

Examples of labile protecting groups include, for example, carboxylateesters, which may hydrolyzed to form an alcohol and a carboxylic acid byexposure to acidic or basic conditions; silyl ethers such as trialkylsilyl ethers, which can be hydrolysed to an alcohol by acid or fluorideion; p-methoxybenzyl ethers, which may be hydrolysed to an alcohol, forexample, by oxidizing conditions or acidic conditions;t-butyloxycarbonyl and 9-fluorenylmethyloxycarbonyl, which may behydrolysed to an amine by a exposure to basic conditions; sulfonamides,which may be hydrolysed to a sulfonate and amine by exposure to asuitable reagent such as samarium iodide or tributyltin hydride; acetalsand ketals, which may be hydrolysed to form an aldehyde or ketone,respectively, along with an alcohol or diol, by exposure o acidicconditions; acylals (i.e., wherein a carbon atom is attached to twocarboxylate groups), which may be hydrolysed to an aldehyde of ketone,for example, by exposure to a Lewis acid; orthoesters (i.e., wherein acarbon atom is attached to three alkoxy or aryloxy groups), which may behydrolysed to a carboxylate ester (which may be further hydrolysed asdescribed hereinabove) by exposure to mildly acidic conditions;2-cyanoethyl phosphates, which may be converted to a phosphate byexposure to mildly basic conditions; methylphosphates, which may behydrolysed to phosphates by exposure to strong nucleophiles; phosphates,which may be hydrolysed to alcohols, for example, by exposure tophosphatases; and aldehydes, which may be converted to carboxylic acids,for example, by exposure to an oxidizing agent.

According to some embodiments of the present invention, a linking moietyis formed as a result of a bond-forming reaction between two (first andsecond) reactive groups.

Exemplary linking moieties, according to some embodiments of the presentinvention, which are formed between a first and a second reactive groupsas described herein include without limitation, amide, lactone, lactam,carboxylate (ester), cycloalkene (e.g., cyclohexene), heteroalicyclic,heteroaryl, triazine, triazole, disulfide, imine, aldimine, ketimine,hydrazone, semicarbazone and the likes. Other linking moieties aredefined hereinbelow.

For example, a reaction between a diene reactive group and a dienophilereactive group, e.g. a Diels-Alder reaction, would form a cycloalkenelinking moiety, and in most cases a cyclohexene linking moiety. Inanother example, an amine reactive group would form an amide linkingmoiety when reacted with a carboxyl reactive group. In another example,a hydroxyl reactive group would form an ester linking moiety whenreacted with a carboxyl reactive group. In another example, a sulfhydrylreactive group would form a disulfide (—S—S—) linking moiety whenreacted with another sulfhydryl reactive group under oxidationconditions, or a thioether (thioalkoxy) linking moiety when reacted witha halo reactive group or another leaving-reactive group. In anotherexample, an alkynyl reactive group would form a triazole linking moietyby “click reaction” when reacted with an azide reactive group.

The “click reaction”, also known as “click chemistry” is a name oftenused to describe a stepwise variant of the Huisgen 1,3-dipolarcycloaddition of azides and alkynes to yield 1,2,3-triazole. Thisreaction is carried out under ambient conditions, or under mildmicrowave irradiation, typically in the presence of a Cu(I) catalyst,and with exclusive regioselectivity for the 1,4-disubstituted triazoleproduct when mediated by catalytic amounts of Cu(I) salts [V.Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. Int.Ed. 2002, 41, 2596; H. C. Kolb, M. Finn, K. B. Sharpless, Angew Chem.,Int. Ed. 2001, 40, 2004].

As demonstrated in the Examples section that follows, the “clickreaction” is particularly suitable in the context of embodiments of thepresent invention since it can be carried out under conditions which arenon-distructive to DNA molecules, and it affords attachment of alebeling agent to 5hmC in a DNA molecule at high chemical yields usingmild conditions in aqueous media. The selectivity of this reactionallows to perform the reaction with minimized or nullified use ofprotecting groups, which use often results in multistep cumbersomesynthetic processes.

In exemplary embodiments, the first and second reactive groups comprise(in no particular order) an azide and an alkyne. These two reactivegroups may combine to form a triazole ring, as defined herein, as alinking moiety. These two reactive groups thus combine to attach alabeling agent to the 5hmC in the DNA molecule by a mechanism referredto as “click” chemistry, as defined herein.

The term “derivatized”, as used herein in the context of a labelingagent and a glucose, means that the labeling agent and/or the glucoseare substituted, or are modified by substituting a position thereof, bya chemical moiety that comprises the respective (first or second)reactive group.

For example, a labeling agent derivatized by a second reactive group, asdescribed herein, means that a labeling agent as described herein ismodified so as to comprise a second reactive group as described herein,by substituting a position thereof with a chemical moiety that comprisesthe second reactive group. Alternatively, the second reactive group or achemical moiety comprising the second reactive group already forms apart in a labeling agent as a substituent.

A chemical moiety that comprises the second reactive group can be thesecond reactive group per se or, for example, a spacer moiety thatincludes, and preferably terminates with, the second reactive group.

As used herein, the phrase “spacer moiety” describes a chemical moietythat typically extends between two chemical moieties and is attached toeach of the chemical moieties via covalent bonds. The spacer moiety maybe linear or cyclic, be branched or unbranched, rigid or flexible.

According to some embodiments of the present invention, the spacermoieties are selected such that they allow and/or promote the one orboth of attachment of a second reactive group to the labeling agent andattachment of the labeling agent to the 5hmC in a DNA molecule. Suchtraits can be selected for in terms of spacer's length, flexibility,structure and specific chemical reactivity or lack thereof.

Exemplary spacer moieties include, but are not limited to, alkyl,alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, heteroaryl and/or ahydrocarbon chain having 1-20 carbon atoms and ending or interrupted byat least one heteroatom selected from the group consisting of O, S and Nand/or containing from 0 to 19 unsaturated carbon-carbon orcarbon-heteroatom bonds.

Additional spacer moieties include, without limitation, —CH₂—, —CH₂—O—,—(CH₂)₂—, —(CH₂)₂—O—, —(CH₂)₃—, —(CH₂)₃—O—, —(CH₂)₄—, —(CH₂)₅—,—(CH₂)₆—, —(CH(CH₃))—CH₂—, —CH═CH—CH═CH—, —C≡C—C≡C—, —CH₂CH(OH)CH₂—,—CH₂—O—CH₂—, —CH₂—O—CH₂—O—, —(CH₂)₂—O—(CH₂)₂—, —(CH₂)₂—O—(CH₂)₂—O—,—CH₂-mC₆H₄—CH₂—, —CH₂-mC₆H₄—CH₂—O—, —CH₂-pC₆H₄—CH₂—, —CH₂-pC₆H₄—CH₂—O—,—CH₂—NHCO—, —C₆H₄—NHCO—, —CH₂—O—CH₂— and —CH═CH—CH₂—NH—(CH₂)₂—, and anycombination thereof. Short polymeric chains, such as, for example,polyalkyelene glycols, are also contemplated.

In exemplary embodiments, a second reactive group as described herein isattached to a labeling agent via a spacer moiety, while exploitingfunctional groups present in the labeling agent for attaching theretothe spacer moiety which terminates with the second reactive group.

A labeling agent derivatized by a second reactive group as describedherein can be selected and prepared using conventional chemicalreactions, or can be a commercially available derivatized labelingagent.

In exemplary embodiments, the second reactive group is an alkyne and thelabeling agent is derivatized by a chemical moiety that comprises analkyne, as described herein. Such a chemical moiety can comprise, forexample, dibezylcyclooctyne (DIBO), and can be attached to the labelingagent via a spacer as described herein.

According to some of these embodiments, the second reactive group is a“strained alkyne”.

A “strained alkyne” is a cycloalkyne, preferably substituted by one ormore groups that render it highly strained, for example, cyclopropyls,benzyls, and others. Examples of known strained alkynes include, but arenot limited to, the following:

The use of a strained alkyl allows performing the click reaction withoutusing a copper catalyst.

A glucose derivatized by a first reactive group describes a glucosemoiety that is substituted at one position thereof by a chemical moietythat comprises the first reactive group, as described herein.

For example, one of the hydroxy groups of a glucose can be substitutedby a chemical moiety that comprises the first reactive group or can beused to attach to the glucose the chemical moiety that comprises thefirst reactive group, via chemical reactions that involve a hydroxygroup, as described herein.

A chemical moiety that comprises the first reactive group can be thefirst reactive group per se or, for example, a spacer moiety, asdescribed herein, that includes, or terminates with, the first reactivegroup.

In exemplary embodiments, one of the hydroxy groups of a glucose issubstituted (replaced) by a chemical moiety that comprises the firstreactive group. Chemical reactions for substituting a hydroxy group arewell known in the art.

In some of these embodiments, the first reactive group is azide and ahydroxy at position 6 of the glucose is substituted by an azide group.

An exemplary synthetic pathway for preparing 6-azido-glucose is depictedin FIG. 1.

According to some embodiments of the invention, a DNA molecule in whichthe 5-hydroxymethylcytosine bases are glycosylated by a glucose moleculederivatized by the first reactive group is prepared, while utilizing aglucose derivatized by the first reactive group, as described herein.

In some embodiments, a selective introduction of a glucose derivatizedby the first reactive group to 5-hydroxymethylcytosines in a DNAmolecule comprises incubating the DNA molecule withβ-glucosyltransferase and a uridine diphosphoglucose (UDP-Glu)derivatized by the first reactive group.

A DNA beta-glucosyltransferase (EC 2.4.1.27) is an enzyme that catalyzesthe chemical reaction in which a beta-D-glucosyl residue is transferredfrom UDP-glucose to an hydroxymethylcytosine residue in DNA. This enzymebelongs to the family of glycosyltransferases, specifically thehexosyltransferases. The systematic name of this enzyme class isUDP-glucose:DNA beta-D-glucosyltransferase. Other names in common useinclude T4-HMC-beta-glucosyl transferase, T4-beta-glucosyl transferase,T4 phage beta-glucosyltransferase, UDP glucose-DNAbeta-glucosyltransferase, and uridine diphosphoglucose-deoxyribonucleatebeta-glucosyltransferase. In certain aspects, the aβ-glucosyltransferase is a His-tag fusion protein.

In other embodiments, the protein may be used without the His-tag(hexa-histidine tag shown above) portion.

A uridine diphosphoglucose (UDP-Glu) derivatized by the first reactivegroup is meant to describe a uridine diphosphoglucose in which theglucose moiety is derivatized by a first reactive group, according toany one of the embodiments described herein.

In some embodiments, the uridine diphosphoglucose (UDP-Glu) derivatizedby the first reactive group is a UDP-6-N₃-Glucose (see, FIG. 2).

A UDP-6-N₃-Glucose, or any other uridine diphosphoglucose (UDP-Glu)derivatized by the first reactive group, can be prepared by chemicalsynthesis, while utilizing, for example, a 6-azido glucose or any otherderivatized glucose, or can be a commercially available product.

In some embodiments, the UDP-6-N₃-Glucose, or any other uridinediphosphoglucose (UDP-Glu) derivatized by the first reactive group, isprepared by enzymatically-catalyzed reactions, as exemplified in furtherdetail hereinafter.

Once a glucose derivatized by a first reactive group is introduced to5-hmCs in a DNA molecule, the DNA molecule is reacted with a labelingagent derivatized by a compatible second reactive group, as describedherein.

As discussed hereinabove, in some embodiments, the reaction involves aclick chemistry reaction.

According to some embodiments of the invention, the click chemistryreaction is free of a copper catalyst, namely, is effected without thepresence of a copper catalyst or any other catalyst that may adverselyaffect the DNA molecule.

For any one of the embodiments described herein throughout, the phrase“labeling agent” refers to a detectable moiety or a probe. Exemplarylabeling agents which are suitable for use in the context of theseembodiments include, but are not limited to, a fluorescent agent, aradioactive agent, a magnetic agent, a chromophore, a bioluminescentagent, a chemiluminescent agent, a phosphorescent agent and a heavymetal cluster, as well as any other known detectable agents.

In some embodiments, the labeling agent is an agent that is detectableby spectrophotometric measurements, and/or which can be utilized toproduce optical imaging. Such agents include, for example, chromophores,fluorescent agents, phosphorescent agents, and heavy metal clusters.

As used herein, the term “chromophore” refers to a chemical moiety that,when attached to another molecule, renders the latter colored and thusvisible when various spectrophotometric measurements are applied.

The phrase “fluorescent agent” refers to a compound that emits light ata specific wavelength during exposure to radiation from an externalsource.

The phrase “phosphorescent agent” refers to a compound emitting lightwithout appreciable heat or external excitation as by slow oxidation ofphosphorous.

A heavy metal cluster can be for example a cluster of gold atoms used,for example, for labeling in electron microscopy techniques (e.g., AFM).

The term “bioluminescent agent” describes a substance which emits lightby a biochemical process.

The term “chemiluminescent agent” describes a substance which emitslight as the result of a chemical reaction.

According to some embodiments of the invention, the labeling agent is afluorescent labeling agent.

A fluorescent agent can be a protein, quantum dots or small molecules.Common dye families include, but are not limited to Xanthenederivatives: fluorescein, rhodamine, Oregon green, eosin, Texas redetc.; Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine,thiacarbocyanine and merocyanine; Naphthalene derivatives (dansyl andprodan derivatives); Coumarin derivatives; oxadiazole derivatives:pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole; Pyrenederivatives: cascade blue etc.; BODIPY (Invitrogen); Oxazinederivatives: Nile red, Nile blue, cresyl violet, oxazine 170 etc.;Acridine derivatives: proflavin, acridine orange, acridine yellow etc.;Arylmethine derivatives: auramine, crystal violet, malachite green; CFdye (Biotium); ALEXA FLUOR® (Invitrogen); Atto and Tracy (SigmaAldrich); FLUOPROBES® (Interchim); Tetrapyrrole derivatives: porphin,phtalocyanine, bilirubin; cascade yellow; azure B; acridine orange;DAPI; Hoechst 33258; lucifer yellow; piroxicam; quinine andanthraginone; squarylium; oligophenylenes; and the like.

Other fluorophores include: Hydroxycoumarin; Aminocoumarin;Methoxycoumarin; Cascade Blue; Pacific Blue; Pacific Orange; Luciferyellow; NBD; R-Phycoerythrin (PE); PE-Cy5 conjugates; PE-Cy7 conjugates;Red 613; PerCP; TruRed; FluorX; Fluorescein; BODIPY-FL; TRITC;X-Rhodamine; Lissamine Rhodamine B; Texas Red; Aliaphycocyanin; APC-Cy7conjugates.

ALEXA FLUOR® dyes (Molecular Probes) include: ALEXA FLUOR® 350, ALEXAFLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXAFLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXAFLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610, ALEXA FLUOR® 633, ALEXAFLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXAFLUOR® 750, and ALEXA FLUOR® 790.

Cy Dyes (GE Heathcare) include Cyt, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 andCy7. Nucleic acid probes include Hoechst 33342, DAPI, Hoechst 33258,SYTOX Blue, ChromomycinA3, Mithramycin, YOYO-1, Ethidium Bromide,Acridine Orange, SYTOX Green, TOTO-1, TO-PRO-1, TO-PRO: Cyanine Monomer,Thiazole Orange, Propidium Iodide (PI), LDS 751, 7-AAD, SYTOX Orange,TOTO-3, TO-PRO-3, and DRAQ5.

Cell function probes include Indo-1, Fluo-3, DCFH, DHR, SNARF.

Fluorescent proteins include Y66H, Y66F, EBFP, EBFP2, Azurite, GFPuv,T-Sapphire, Cerulean, mCFP, ECFP, CyPet, Y66W, mKeima-Red, TagCFP,AmCyanl, mTFP1, S65A, Midoriishi Cyan, Wild Type GFP, S65C, TurboGFP,TagGFP, S65L, Emerald, S65T (Invitrogen), EGFP (Ciontech), Azami Green(MBL), ZsGreenl (Clontech), TagYFP (Evrogen), EYFP (Clontech), Topaz,Venus, mCitrine, YPet, Turbo YFP, ZsYellow1 (Clontech), Kusabira Orange(MBL), mOrange, mKO, TurboRFP (Evrogen), tdTomato, TagRFP (Evrogen),DsRed (Clontech), DsRed2 (Clontech), mStrawberry, TurboFP602 (Evrogen),AsRed2 (Clontech), mRFP1, J-Red, mCherry, HcRed1 (Clontech), Katusha,Kate (Evrogen), TurboFP635 (Evrogen), mP!um, and mRaspberry.

It is to be noted that, in some embodiments, each of the labeling agents(e.g., fluophores) is attached to the DNA molecule by means of clickchemistry and that the reagents used for the reaction are derivatives ofthe labeling agent, which include a reactive group as described herein.

Exemplary fluorescent agents include, but are not limited to, ALEXAFLUOR@dyes, Cy Dyes, Atto dyes, TAMRA dyes, etc., such as, for example,described in the Examples section that follows.

According to some embodiments of the invention, analyzing 5hmC contentis done without subjecting the DNA molecule to fragmentation.

As mentioned, the DNA molecule is immobilized on a solid phase.

According to some embodiments of the invention, the extending islinearly extending.

According to some embodiments of the invention, the extending iseffected by depositing the DNA molecule on a surface or extending theDNA molecule in a nanochannel.

As used herein “extended DNA molecule” or “elongated DNA molecule” whichis interchangeably used herein refers to a single or plurality elongatedand fixed (i.e., immobilized) DNA.

According to some embodiments of the invention, the extended DNAmolecules are elongated and fixed in a controllable manner directly ontoa solid, planar surface. According to a specific embodiment, this solid,planar surface contains a positive charge density which has beencontrollably modified such that the single nucleic acid molecules willexhibit an optimal balance between the critical parameters of nucleicacid elongation state, degree of relaxation stability and biologicalactivity. Further, methods, compositions and assays are described bywhich such an optimal balance can precisely and reproducibly beachieved.

According to alternative or additional embodiments, the single nucleicacid molecules are elongated via flow-based techniques. In such anembodiment, a single nucleic acid molecule is elongated, manipulated(via, for example, a regio-specific restriction digestion), and/oranalyzed in a laminar flow elongation device. Such a laminar flowelongation devices and methods of elongating or extending DNA aredescribed in U.S. Patent Application 20030124611, which is herebyincorporated by reference in its entirety.

The elongated, individual labeled DNA molecules can then be utilized ina variety of ways which have applications for the analysis of nucleicacid at the genome level. For example, such nucleic acid molecules maybe used to generate ordered, high resolution single nucleic acidmolecule restriction maps. This method is referred to herein as “opticalmapping” or “optical restriction mapping”. Additionally, methods arepresented whereby specific nucleotide sequences present within theelongated nucleic acid molecules can be identified. Such methods arereferred to herein as “optical sequencing”. The optical mapping andoptical sequencing techniques can be used independently or incombination on the same individual nucleic acid molecules.

Additionally, methods are also presented for the imaging and sizing ofthe elongated single nucleic acid molecules. These imaging techniquesmay, for example, include the use of fluorochromes, microscopy and/orimage processing computer software and hardware.

Further description of DNA extension is provided hereinbelow and in theExamples section which follows.

According to some embodiments of the invention, step (b, extending) iseffected following step (a, attaching to the DNA molecule a 5hmcspecific labeling agent). However, it will be appreciated that extendingthe DNA molecule can be done prior to step (a).

According to some embodiments of the invention, the method furthercomprises attaching to the DNA molecule an additional labeling agentdistinct of the 5hmc specific labeling agent.

According to some embodiments of the invention, the additional labelingagent is an epigenetic modification specific labeling agent. Examples ofsuch modifications include but are not limited to 5-methylcytosine(5mC), histone acetylation and the like.

According to some embodiments of the invention, the additional labelingagent is a non-epigenetic modification specific labeling agent. Examplesof such stains and dyes include DNA fluorescent dyes such as cyaninenucleic acid stains, which are essentially nonfluorescent in the absenceof nucleic acids and exhibit significant fluorescence enhancements uponDNA binding. The stain may be cell permeant or impermeant.

Such stains are available from Molecular Probes (e.g., YOYO-1. TOTO,SYTOX, POPO-1, BOBO-1, LOLO-1, JOJO-1 etc.). Alternatively,non-fluorescent stains can be used as further described hereinbelow.

Still further, high throughput methods for utilizing such single nucleicacid molecules in genome analysis are presented. In one embodiment ofsuch high throughput methods, rapid optical mapping approaches aredescribed for the creation of high-resolution restriction maps. In suchan embodiment, single nucleic acid molecules are elongated, fixed andgridded to high density onto a solid surface. These molecules can thenbe digested with appropriate restriction enzymes for the mapconstruction. In an alternative embodiment, the single nucleic acidmolecules can be elongated, fixed and gridded at high density onto asolid surface and utilized in a variety of optical sequencing-baseddiagnostic methods. In addition to speed, such diagnostic grids can bereused. Further, the high throughput and methods can be utilized torapidly generate information derived from procedures which combineoptical mapping and optical sequencing methods.

According to an aspect of some embodiments of the present inventionthere is provided a method of in-situ imaging a DNA molecule, the methodcomprising:

(a) attaching a labeling agent to the DNA molecule as described herein;and(b) subjecting the DNA molecule to an imaging method suitable fordetecting the labeling agent.

According to some embodiments of the invention, the labeling agent is afluorescent agent, as described herein, and the imaging method is afluorescence imaging.

Other labeling agents, as described herein, are also contemplated andrespective imaging methods are utilized accordingly.

According to some embodiments of the invention, the method furthercomprises generating an optical image of the DNA molecule following theimaging.

According to an aspect of some embodiments of the present inventionthere is provided an extended DNA molecule comprising at least one5hmc-specific labeling agent.

According to an aspect of some embodiments of the present inventionthere is provided a DNA molecule comprising at least two differentlabeling agents, wherein a first labeling agent of the at least twodifferent labels is a 5hmc-specific labeling agent.

According to some embodiments of the invention, the 5hmc-specificlabeling agent is attached to the DNA molecule by reacting a labelingagent derivatized by a second reactive group with a DNA molecule inwhich the 5-hydroxymethylcytosines are glycosylated by a glucosemolecule derivatized by a first reactive group,

wherein the first and second reactive groups are chemically compatibleto one another, as described in any one of the embodiments pertaining toattaching a 5hmc-specific labeling agent to a DNA molecule of thepresent invention.

According to some embodiments of the invention, one the first and secondreactive groups is azide and the other is alkyne, such that attachingthe labeling agent to the DNA molecule is effected by a click chemistry,as described herein.

According to some embodiments of the invention, a second labeling agentof the at least two different labeling agents is a 5mc-specific labelingagent.

According to some embodiments of the invention, a second labeling agentof the at least two different labeling agents is for an epigeneticmodification.

According to some embodiments of the invention, a second labeling agentof the at least two different labeling agents is for anon-epigenetically modified base.

As used herein “distinct” or “different” labels refer to labels whichcan be distinguished upon visualization. Thus in fluorescence labelingone label may be red fluorescence while the other can be bluefluorescence.

According to some embodiments of the invention, the DNA molecule isextended.

According to an aspect of some embodiments of the present inventionthere is provided a composition-of-matter comprising the DNA molecule.

According to some embodiments of the invention, the DNA molecule issurface deposited or extended in a microchannel.

The present invention also envisages detecting 5hmC in non-immobilizedbiological samples.

Thus, according to an aspect of some embodiments of the presentinvention there is provided a method of detecting5-hydroxymethyl-cytosine (5hmC) in a DNA sample the method comprising:

(a) reacting the DNA sample with a 5hmc-specific fluorescent agent underconditions which allow staining of the DNA sample with said5hmc-specific labeling agent so as to obtain a 5hmC-labeled DNA sample;and(b) measuring fluorescence intensity of said 5hmC-labeled DNA sample (X)and absorption intensity of the DNA, at 260 nm (Y), wherein a ratiobetween X to Y is indicative of presence or level of 5hmC in the DNAsample.

As used herein the term “fluorescence intensity” refers to the intensityof the fluorescent probe.

It will be appreciated that for cyanine staining, changes in probeconcentration, by dilution of the sample, for example, can, influencethe fluorescence intensity of the DNA due to the change in equilibrium.For this reason, DNA preparations are typically not washed to removeunbound probe; otherwise the equilibrium will be interrupted. It will beappreciated that the unbound probe typically does not fluoresce and,hence, demonstrates low background fluorescence. The intensity ismeasured at an excitation and emission values which depend on the probe.

As used herein “absorbance” refers to DNA light absorbance at 260 nmwhich is a measure for DNA quantity. At this wavelength, DNA typicallyexhibits absorbance maxima.

According to a specific embodiment, the ratio is compared to aratiometric calibration curve.

The calibration curve can be generated by using DNA samples of knownpercentage of 5hmC labeled using the same methodology as the test DNAsample.

The methodology described herein, according to some embodiments of thepresent invention can be used to detect global 5hmC modification.

As used herein “global 5hmC modification” refers to the detection of5hmC of a plurality of DNA molecules which are in a non-immobilizedstate. The sample may be a heterogeneous sample.

According to a specific embodiment, this methodology is more sensitivethan absorption measurement of labeled 5hmC. Thus as shown in Example 2of the Examples section which follows, measuring the ratio between thefluorescence signal of labeled 5hmC and the absorption of DNA at 260 nmallowed to detect down to 0.004% 5hmC/dN from a sample extracted fromliver, with a sample concentration of 136 ng/μl in 20 μl volume and0.02% 5hmC/dN from a DNA sample concentration of only 82 ng/μl in 20 μlvolume (1.6 μg), see FIGS. 5A-5B.

It is contemplated that the threshold of sensitivity or the limit ofdetection is about 0.0022% 5hmC/dN.

The concentration of the DNA in the test sample depends on the level(e.g., %) of hmC in the tissue. Thus, a higher DNA concentration isrequired for tissues with lower levels hmC. In general when assayedusing a plate reader, the concentration of DNA that can be read is up to350 ng/μl DNA without having signal saturation (e.g., 1-350 ng/μl). Thisconcentration of DNA is high enough for detection % hmC at low-%hmC-containing tissues such as spleen and liver. However for tissuescontaining even lower % hmC, concentrated DNA samples (e.g., 100 ng/μlto 100 μg/μl) may be measured for their fluorescence intensity and thendiluted for measuring their DNA concentration. 1 pg-/μl-100 μg/μl, 1pg/μl-50 μg/μl, e.g., 5 ng/μl-5 μg/μl.

According to a specific embodiment, the volume of the sample is between5-50 μl or 10-20 ul for the detection of hmC in genomic DNA in multiwell plate.

Once the level of 5hmC modification has been determined, the sample canbe subjected to optical imaging by extending the molecules on slides(immobilizing the DNA molecules) as described herein. Alternatively, theposition of the modification can be analyzed using enzymes which aresensitive to bulky residues i.e., the modification of the 5hmC withN₃-5-gmC.

Presence of N₃-5-g group on the DNA template strand will interfere withthe synthesis of a nucleic acid strand by DNA polymerase or RNApolymerase, or the efficient cleavage of DNA by a restrictionendonuclease (e.g., Msp1) or inhibition of other enzymatic modificationsof nucleic acid containing 5-hmC. As a result, primer extensions orother assays can be employed, for example, to evaluate a partiallyextended primer of certain length and the modification sites can berevealed by sequencing the partially extended primers.

The ability to sensitively and specifically detect 5hmC modificationscan be harnessed for large scale settings in which hundreds (e.g.,300-5000) or thousands of DNA samples are analyzed using an automatedequipment.

In certain aspects, differential modification of nucleic acid betweentwo or more samples can be evaluated.

In such differential analysis studies, global DNA samples of differenttissues, age, gender, medical conditions (diseased vs healthy) can beanalyzed.

Alternatively, specific DNA sequences of the aforementioned types can beanalyzed.

Studies including heart, liver, lungs, kidney, muscle, testes, spleen,and brain indicate that under normal conditions 5-hmC is predominatelyin normal brain cells. Additional studies have shown that 5-hmC is alsopresent in mouse embryonic stem cells. The Ten-eleven translocation 1(TET1) protein has been identified as the catalyst for converting 5-mCto 5-hmC. Studies have shown that TET1 expression is inverselycorrelated to 5-mC expression. Overexpression of TET1 in cells seems tocorrelate with increased expression of 5-hmC. Also, TET1 is known to beinvolved in pediatric and adult acute myeloid leukemia and acutelymphoblastic leukemia. Thus, evaluating and comparing 5-hmC levels canbe used in evaluating various disease states and comparing variousnucleic acid samples.

Thus the output of such methods can be used in a variety of research andclinical applications such as in diagnostics, therapy and drugdevelopment.

According to an additional aspect of the present invention there isprovided a computer readable storage medium comprising a databaseincluding a plurality of DNA sequences and information pertaining to5hmC modification of the plurality of DNA molecules.

Thus, the computer readable storage medium may comprise informationpertaining to the position of the 5hmC modification on the DNA sequence,the level of 5hmC, the tissue distribution of a given 5hmC modificationon a DNA molecule. The database further includes information pertainingto the DNA sequence such as annotation, transcribed/translatedmRNA/protein sequence, post translational modifications and the like.

According to still further features in the described preferredembodiments the database further includes information pertaining togeneration of the database and potential uses of the database.

According to an aspect of some embodiments of the present inventionthere is provided a method of preparing UDP-6-N₃-Glucose, the methodcomprising subjecting an azido glucose (6-azido glucose) an enzymaticcatalysis by kinase N-acetylhexoseamine 1-kinase (NahK), in the presenceof ATP to thereby obtain a phosphorylated 6-azidoglucose; and subjectingthe phosphorylated 6-azido glucose to enzymatic catalysis byuridyltransferase (GlmU) in the presence of UTP.

According to an aspect of some embodiments of the present inventionthere is provided a method of preparing UDP-6-N₃-Glucose, as depicted inFIG. 1.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

The phrase “covalent bond”, as used herein, refers to one or more pairsof electrons which are shared between atoms in a form of chemicalbonding.

The term “amide” describes a —NR′—C(═O)— linking moiety, where each ofR′ and R″ is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic,aryl or heteroaryl, as these terms are defined herein.

The term “carboxylate” or “ester”, as used herein, refers to a—C(═O)—O—R′ end group, where R′ is as defined herein, or a—C(═O)—O—linking moiety.

The term “triazine” refers to a heterocyclic ring, analogous to thesix-membered benzene ring but with three carbons replaced by nitrogenatoms. The three isomers of triazine are distinguished from each otherby the positions of their nitrogen atoms, and are referred to as1,2,3-triazine, 1,2,4-triazine, and 1,3,5-triazine. Other aromaticnitrogen heterocycles include pyridines with 1 ring nitrogen atom,diazines with 2 nitrogen atoms in the ring and tetrazines with 4 ringnitrogen atoms.

The term “triazole” refers to either one of a pair of isomeric chemicalcompounds with molecular formula C₂H₃N₃, having a five-membered ring oftwo carbon atoms and three nitrogen atoms, namely 1,2,3-triazoles and1,2,4-triazoles.

The term “disulfide” refers to a —S—S— linking moiety.

The term “imine”, which is also referred to in the art interchangeablyas “Schiff-base”, describes a —N═CR′— linking moiety, with R′ as definedherein or hydrogen. As is well known in the art, Schiff bases aretypically formed by reacting an aldehyde or a ketone and anamine-containing moiety such as amine, hydrazine, hydrazide and thelike, as these terms are defined herein. The term “aldimine” refers to a—CH═N— imine which is derived from an aldehyde. The term “ketimine”refers to a —CR′═N— imine which is derived from a ketone.

The term “hydrazone” refers to a —R′C═N—NR″ linking moiety, wherein R′and R″ are as defined herein.

The term “semicarbazone” refers to a linking moiety which forms in acondensation reaction between an aldehyde or ketone and semicarbazide. Asemicarbazone linking moiety stemming from a ketone is a—R′C═NNR″C(═O)NR′″—, and a linking moiety stemming from an aldehyde is a—CR′═NNR″C(═O)NR′″ wherein R′ and R″ are as defined herein and R′″ or asdefined for R′.

As used herein, the term “lactone” refers to a cyclic ester, namely theintra-condensation product of an alcohol group —OH and a carboxylic acidgroup —COOH in the same molecule.

As used herein, the term “lactam” refers to a cyclic amide, as this termis defined herein. A lactam with two carbon atoms beside the carbonyland four ring atoms in total is referred to as a β-lactam, a lactam withthree carbon atoms beside the carbonyl and five ring atoms in total isreferred to as a γ-lactam, a lactam with four carbon atoms beside thecarbonyl and six ring atoms in total is referred to as a δ-lactam, andso on.

As used herein, the term “aldehyde” refers to an —C(═O)—H group.

The term “hydroxy” as used herein describes an —OH group.

The terms “thio”, “sulfhydryl” or “thiohydroxy” as used herein describean —SH group.

The term “disulfide” as used herein describes an —S—S— linking moiety.

The term “alkoxy” as used herein describes an —O-alkyl, an—O-cycloalkyl, as defined hereinabove. The ether group —O— is also apossible linking moiety.

The term “aryloxy” as used herein describes an —O-aryl group.

The term “thioalkoxy” as used herein describes an —S-alkyl group. Thethioether group —S— is also a possible linking moiety.

The term “thioaryloxy” as used herein describes an —S-aryl group. Thethioarylether group —S-aryl- is also a possible linking moiety.

As used herein, the term “amine” refers to an —NR′R″ group where R′ andR″ are each hydrogen, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl(bonded through a ring carbon) or heteroalicyclic (bonded through a ringcarbon) as defined hereinbelow.

The terms “halide” or “halo” refer to fluorine, chlorine, bromine oriodine.

As used herein, the term “azide” refers to a —N₃ (—N═N⁺═N⁻) group.

The term “aziridine”, as used herein, refers to a reactive group whichis a three membered heterocycle with one amine group and two methylenegroups, having a molecular formula of —C₂H₃NH.

The term “diene”, as used herein, refers to a —CR′═CR″—CR′″═CR″″ group,wherein R′ as defined hereinabove, and R″ R′″ and R″″ are as defined forR′.

The term “dienophile”, as used herein, refers to a reactive group thatreacts with a diene, typically in a Diels-Alder reaction mechanism,hence a dienophile is typically a double bond or an alkenyl.

The term “epoxy”, as used herein, refers to a reactive group which is athree membered heterocycle with one oxygen and two methylene groups,having a molecular formula of —C₂H₃O.

The term “azo” or “diazo” describes an —N═NR′ reactive group or an—N═N-linking moiety, as these phrases are defined hereinabove, with R′as defined hereinabove.

The term “carbamate” refers to a —NR′(C═O)OH (carbamic acid) end orreactive group, or a —NR′(C═O)O— linking moiety, with R′ as definedhereinabove.

The term “thiocarbamate” refers to a —NR′(C═S)OH end or reactive group,or a —NR′(C═S)O— linking moiety, with R′ as defined hereinabove.

The term “carbonyl” refers to a —(C═O)— group.

The term “thiocarbonyl” refers to a —(C═S)— group.

As used herein, the term “carboxyl” refers to an —C(═O)OH group.

The term “cyano” describes a —C≡N group.

The term “isocyanate” describes an —N═C═O group.

The term “hydrazide”, as used herein, refers to a —C(═O)—NR′—NR″R′″group wherein R′, R″ and R′″ are each independently hydrogen, alkyl,cycloalkyl or aryl, as these terms are defined herein.

As used herein, the term “hydrazine” describes a —NR′—NR″R′″ group,wherein R′, R″ and R′″ are each independently hydrogen, alkyl,cycloalkyl or aryl, as these terms are defined herein.

The term “hydroxylamine”, as used hereon, refers to either a —NHOH groupor a —ONH₂.

The term “nitro” describes an —NO₂ group.

The term “acyl halide” describes a —(C═O)R″″ group wherein R″″ ishalide, as defined hereinabove.

The term “phosphate” describes an —O—P(═O)₂(OR′) end or reactive groupor a —O—P(═O)₂(O)— linking moiety, as these phrases are definedhereinabove, with R′ as defined herein.

The term “phosphonate” describes a —P(═O)(OR′)(OR″) end or reactivegroup or a —P(═O)(OR′)(O)— linking moiety, as these phrases are definedhereinabove, with R′ and R″ as defined herein.

The term “sulfoxide” or “sulfinyl” describes a —S(═O)R′ end or reactivegroup or an —S(═O)— linking moiety, as these phrases are definedhereinabove, where R′ is as defined hereinabove.

The term “sulfonamide” encompasses the term “S-sulfonamide” whichdescribes a —S(═O)₂—NR′R″ end or reactive group or a —S(═O)₂—NR′—linking moiety, as these phrases are defined hereinabove, with R′ and R″as defined herein; and the term “N-sulfonamide” which describes anR'S(═O)₂—NR″— end or reactive group or a —S(═O)₂—NR′— linking moiety, asthese phrases are defined hereinabove, where R′ and R″ are as definedherein.

The term “sulfonate” describes a —S(═O)₂—R′ end or reactive group or an—S(═O)₂— linking moiety, as these phrases are defined hereinabove, whereR′ is as defined herein.

As used herein, the term “alkyl” describes an aliphatic hydrocarbonincluding straight chain and branched chain groups. Preferably, thealkyl group has 1 to 20 carbon atoms, and more preferably 1-10 carbonatoms. Whenever a numerical range; e.g., “1-10”, is stated herein, itimplies that the group, in this case the alkyl group, may contain 1carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including10 carbon atoms. The alkyl can be substituted or unsubstituted. Whensubstituted, the substituent can be, for example, an alkyl, an alkenyl,an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a halide, a hydroxy, analkoxy and a hydroxyalkyl as these terms are defined hereinbelow.

The term “alkenyl” describes an unsaturated alkyl, as defined herein,having at least two carbon atoms and at least one carbon-carbon doublebond. The alkenyl may be substituted or unsubstituted by one or moresubstituents, as described for alkyl hereinabove.

The terms “alkynyl” or “alkyne”, as defined herein, is an unsaturatedalkyl having at least two carbon atoms and at least one carbon-carbontriple bond. The alkynyl may be substituted or unsubstituted by one ormore substituents, as described hereinabove.

The term “cycloalkyl” describes an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereone or more of the rings does not have a completely conjugatedpi-electron system. The cycloalkyl group may be substituted orunsubstituted as described for alkyl hereinabove.

The term “heteroalicyclic” describes a monocyclic or fused ring grouphaving in the ring(s) one or more atoms such as nitrogen, oxygen andsulfur. The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system. Theheteroalicyclic may be substituted or unsubstituted as described foralkyl hereinabove. Representative examples are piperidine, piperazine,tetrahydrofurane, tetrahydropyrane, morpholino and the like.

The term “aryl” describes an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. The aryl groupmay be substituted or unsubstituted. Substituted aryl may have one ormore substituents as described for alkyl hereinabove.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted as described for alkyl hereinabove.Representative examples of heteroaryls include triazole, furane,imidazole, indole, isoquinoline, oxazole, pyrazole, pyridine,pyrimidine, pyrrole, quinoline, thiazole, thiophene, triazine, purineand the like.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 5hmC Labeling Material and Experimental Methods

Synthesis of UDP-6-azide Glucose:

The T4 bacteriophage uses the enzyme β-glucosyltransferase (β-GT) toglucosilate hydroxylated bases in its DNA. Recently, a selectivechemical method for the labeling of 5hmC was developed. In this methodβ-GT was exploited in order to transfer an azide-substituted glucose(UDP-6-N₃-Glu) onto the hydroxyl group of 5hmC to formβ-6-azide-glucosyl-5-hydroxymethyl-cytosine (5-N₃-gmC). The presence ofan azido moiety paves the way for 5hmC labeling using a click chemistryreaction by the addition of an alkyne substituted dye.

The main bottleneck of this labeling process is the cofactorUDP-6-N₃-Glu, which can be synthesized or purchased but with very highcosts.

Herein, an enzymatic approach is used in order to have high yields ofUDP-N₃-Glu. A glucose azide (see FIG. 1) is used as a substrate for asequential enzymatic cascade.

The glucose azide can be synthesized as described in FIG. 1, and then besubjected to the following reactions: a) First, the kinaseN-acetylhexoseamine 1-kinase (NahK) is used to add a phosphate group tothe glucose, using ATP as a co-factor and b) Second, anuridyltransferase (GlmU) is used with UTP as a cofactor to catalyzeconjugation of a UDP group to form the desired UDP-N₃-Glu.

NahK and GlmU are purified as discussed previously (Chen et al., 2011).

DNA Samples:

5hmC-saturated DNA fragments of 1 kb and 70 bp were prepared by PCRamplification of lambda DNA (New England Biolabs; (NEB), Ipswich Mass.,USA), using the following primers: forward primer:5-CTCATGCTGAAAACGTGGTG-3 (SEQ ID NO: 1), reverse primer:5-GGACAGGACCAGCATACGAT-3 (SEQ ID NO: 2) and forward primer:5-/5Alex488N/TAAATTAGTTACACAGGAAA-3 (SEQ ID NO: 3) reverse primer:5-AAGCCACAA CTCTAATTTT-3 (SEQ ID NO: 4) for 1 kb and 70 bp DNAfragments, respectively (Integrated DNA Technologies Inc, Coralville,Iowa USA). A typical reaction was performed in a volume of 50 μl, andcontained 200 ng of template DNA, 2 units of Vent (exo-) (NEB), 200 μMof dAGT (Sigma-Aldrich Israel Ltd. Rehovot, Israel) and 5hmC (BiolineReagents Ltd., London, UK) nucleotides in NEB thermopol buffer. Reactionmixtures were incubated at 95° C. for 2 minutes as an initial step,followed by 30-35 cycles of 30 seconds at 95° C., 30 seconds at 55° C.and 2 minutes at 72° C. or 30 seconds at 95° C., 30 seconds at 42° C.and 30 seconds at 72° C. for the 1 kb and 70 bp products, respectively,and finally 5 minutes at 72° C.

For the analysis of the labeling efficiency of 5 hmC, the 70 bp lambdaDNA fragments were prepared with ALEXA FLUOR® 647-dCTP (MolecularProbes, Eugene, Oreg., USA) instead of 5hmC. The reaction was performedin a volume of 50 μl, and contained 200 ng of template DNA, 2 units ofVent (exo-), 50 μM of dATP dGTP and dTTP and 50 μM ALEXA FLUOR® 647-dCTPnucleotides in NEB thermopol buffer. Reaction mixtures were incubated at95° C. for 2 minutes as an initial step, followed by 35 cycles of 30seconds at 95° C., 30 seconds at 42° C. and 30 minutes at 50° C., andfinally 10 minutes at 72° C. This control ALEXA FLUOR® 647-PCR productcontained three cytosine sites and represents 100% labeling efficiency.All PCR products were cleaned of free nucleotides and primers usingQIAquick PCR purification kit (QIAGEN GmbH, Hilden, Germany).

For extraction of DNA from mouse tissues, 5 prime ArchivePure DNAcell/tissue kit was used according to manufacturer's instructions.

Preparation of Hydroxymethylated Lambda DNA by Sequence SpecificLabeling:

Lambda phage intact genomes (48.5 kb) were labeled with 5hmC nucleotidesby nick translation^([1]). 10 μg of DNA was incubated with 10 u/μgNt.BspQI (NEB) nicking enzyme in 100 μl NEB buffer 3 for 2 h at 50° C.,followed by heat inactivation for 20 min at 80° C. For labeling of theDNA, nicked DNA was incubated for 2 h at 72° C. in 200 μl NEB thermopolbuffer with 2 u/μg Vent (exo-) and 250 nM dNTPs. For incorporation ofhydroxymethyl, DNA was incubated with 250 nM dATP dGTP and dTTP and 250nM of 5-hydroxymethyl-labeled cytosine (Bioline).

Fluorescent-Labeling of 5hmC by Click Chemistry:

In the case of lambda DNA for single molecule optical mapping, 10 μg ofNt.BspQI site Hydroxymethylated DNA in 50 mM Hepes (Sigma-Aldrich IsraelLtd.) was incubated with 20 units of T4-beta-glucosyltransferase (NEB)for glucosylation of 5hmC, in the presence of NEB buffer 4 and 150 μMUDP-azide glucose (Active Motif, Carlsbad, Calif., USA), for 2 hours at37° C. As a control reaction, UDP-azide glucose was replaced byUDP-glucose at the same molar concentration. The click chemistryreaction was performed, copper-free, by the addition of 250 μM ALEXAFLUOR® 555 DIBO alkyne (Molecular Probes) for 1 hour at 37° C. Bufferwas then exchanged to 50 mM Hepes, and sample was stained with 1 μMYOYO-1.

For labeling 5hmC-saturated PCR products, 1-3 μg was first glucosylatedby incubation with UDP-glucose-azide (Active Motif) at a molar ratio of1:30 (5hmC: UDP-glucose-azide) and 50 units ofT4-beta-glucosyltransferase (NEB), in the presence of NEB buffer 4,overnight at 37° C. Two types of click reactions were used: First, acopper-free reaction, with ALEXA FLUOR® 647-DIBO (dibenzocyclooctyne)alkyne (Molecular Probes) (for the 1 kb PCR product) at a molar ratio of1:100 (5hmC: DIBO) in 10 mM PBS. Second, a copper-dependent reactionwith ALEXA FLUOR® 647-alkyne at a molar ratio of 1:100 (5hmC: alkyne) inthe presence of 200 mM triethylammonium acetate buffer, 50% DMSO,freshly prepared 0.5 mM ascorbic acid and 0.5 mM Cu-TBTA(Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine complex in 55% DMSO.The copper in the second reaction served as a catalyst which was notrequired when using cyclooctynes as in the DIBO alkyne. Both reactionswere incubated at 22° C. overnight. The reaction sample was degassed bynitrogen before addition of Cu-TBTA complex and flashed beforeincubation. As a control reaction, UDP-glucose- azide was replaced byUDP-glucose at the same molar concentration.

Each incubation step preceded a purification step with Qiagen PCRpurification columns (QIAGEN).

Using the synthetic pathways described hereinabove, click chemistrysyntheses were performed for labeling 5-hmC, using the followingalkyne-containing fluofhores:

DBCO-Cy5 (Dibenzylcyclooctyne-Sulfo-Cy5, Gena Bioscience); BCN Cy5 [N.J. Agard, J. A. Prescher and C. R. Bertozzi, J. Am. Chem. Soc., 2004,126, 15046; (b) N. J. Agard, J. M. Baskin, J. A. Prescher, A. Lo and C.R. Bertozzi, ACS Chem. Biol., 2006, 1, 644; (c) J. M. Baskin, J. A.Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang, I. A. Miller, A. Lo,J. A. Codelli and C. R. Bertozzi, Proc. Natl. Acad. Sci. U.S.A, 2007,104, 16793; (d) S. T. Laughlin, J. M. Baskin, S. L. Amacher and C. R.Bertozzi, Science, 2008, 320, 664; (e) E. M. Sletten and C. R. Bertozzi,Org. Lett., 2008, 10, 3097, BCN-amine was purchased from Sigma-AldrichCat #745073 and coupled cy5-NHS ester, Fischer Scientific Cat#45-001-190]; DBCO-TAMRA(Dibenzylcyclooctyne-PEG₄-5/6-Tetramethylrhodamine, Gena Bioscience).

5hmC Quantification by UV-Vis Spectroscopy:

In order to determine the percentage of 5hmC in an examined DNA sample,a calibration curve was obtained with a DNA sample that contains a known5hmC percentage. For this purpose, ALEXA FLUOR® 647-5hmC labeled-1 kbPCR product was mixed with increasing concentrations of non-labeled XL1bacteria plasmids and the absorption ratio at 260 nm for DNA and 647 nmfor labeled 5hmC was plotted. Increasing amounts of plasmids (600, 1200and 1750, 3500 ng) were added to fixed amounts of 5hmC-saturated 1 kblambda fragments (in the order of 4 and 2 ng). The 1 kb fragmentcontained 29% 5hmC and was fluorescently labeled by the click reaction.Plasmids were extracted from a XL1 PE bacteria by a DNA purificationsystem (Promega, Madison Wis., USA). Absorption measurements wereconducted on a NanoPhotometer® P 300 (IMPLEN, Munich, Germany).

Analysis of Labeling Efficiency:

ALEXA FLUOR® 488-labeled-70 bp PCR products that were prepared withALEXA FLUOR® 647-dCTP nucleotides, or with 5hmC nucleotides, were used.The 5hmC product was subjected to click reaction and the two sets of DNAsamples were analyzed by electrophoresis and run side by side through a3% agarose gel, in TBE buffer, at 80 volts. The gel was imaged on amulticolour laser gel scanner, GE Healthcare FLA5000. A normalization ofthe DNA amount loaded on the gel was achieved by comparing thefluorescence intensity of the 70 bp bands at 510 nm, resulting from theALEXA FLUOR® 488 molecule bound to the forward primer of both controland click reaction products, following excitation with 473 nm laser. Theefficiency of the click labeling procedure was calculated by comparingthe bands fluorescence intensity at 665 nm, resulting from the ALEXAFLUOR®-labeled dC nucleotides or 5hmC subjected to a click reaction,following excitation with 635 nm laser. Fluorescence intensitymeasurements were analyzed by ImageJ:www(dot)rsbweb(dot)nih(dot)gov/ij/.

DNA Extension:

Surfaces for DNA extensions were prepared according to Sidorova etal.^([2]) with minor modifications. Briefly, 24×24 glass cover slipswere cleaned by 7 hours to overnight incubation in a freshly made 2:1(v/v) mixture of nitric acid (70%) and hydrochloric acid (37%). Theincubation proceeded in a chemical fume hood and was followed by anextensive wash with ultrapure water (18 MΩ), ethanol and dried under astream of nitrogen. Dry slides were immersed in a premixed solutioncontaining 595 μl N-trimethoxysilylpropyl-N,N,N-trimethylammoniumchloride and 216 μl of vinyltrimethoxysilane in 300 ml ultrapure waterand incubated overnight at 65° C. After incubation, slides werethoroughly washed with ultrapure water and ethanol and stored at 4° C.in ethanol. The silane solution was freshly made and thoroughly mixedbefore the slides were introduced into the mixture. Stored slides werenormally used within 2 weeks. Lambda DNA molecules were extended onsilanized glass slides by placing a 5 μl drop of pre-labeled Lambda DNAin 50 mM HEPES buffer and 200 mM dithiothreitol (DTT) in-between a drysilanized glass slide and a non-treated microscope glass slide (lambdaDNA concentration was as appropriate for single molecule imaging).

Data Acquisition and Analysis:

Extended DNA molecules were imaged on a MORE imaging system (TILLphotonics GmbH, Munich, Germany) with an Olympus UPlanApo 60X 1.35 NAoil immersion objective. A 150 W Xenon lamp with galvanometer drivenfilter switching was used as an excitation source. The filter sets usedto image YOYO-1 stained DNA and the ALEXA FLUOR® 555 labels were 482/18and 561/14 bandpass excitation filters, 405/488/561/640 quadbandbeamsplitter and a 446/523/600/677 quadband emission filter (all fromSemrock, Rochester, N.Y., USA). Images were acquired by a DU888 EMCCD(Andor, Belfast, Ireland) with an EM gain setting of 200 and integrationtimes of 200 ms and 1300 ms for YOYO-1 and ALEXA FLUOR® 555respectively.

The positions of the ALEXA FLUOR® 555 fluorescence spots along the DNAwere mapped in order to verify that they are located in the expectedpositions of 5hmC sites. In order to determine the genomic position ofthe tags the distance (in pixels) between the Tag signal position andthe far end of the DNA was measured. This value was divided by themeasured full length of the DNA to give a normalized position value(ranging from 0 to 100). Fluorescence spots were mapped manually using aMatlab program written for this purpose. The spectrally separated imagesof the stretched DNA and of the ALEXA FLUOR® 555 fluorescent spots wereoverlaid to visualize fluorescent tags bound on the hydroxymethylatedDNA bases. Using the Matlab function Improfile, a line was manuallydrawn along each long DNA strand (>80 pixels) that showed at least fourfluorescent spots along its contour and had orientation that was clearlyevident from the spot pattern. The Improfile function projects theintensity values of each pixel along the drawn line on a pixel vs.intensity plot. The DNA lengths were measured by subtracting the twoy-intercepts on the DNA channel that represent DNA ends. Fluorescencespots generated by labeled 5hmC bases may be accurately localized by 2DGaussian fittingE^([3,4]). Images were analyzed using a custom Matlabprogram that extracts the position coordinates of fluorescent spots.After localizing each spot, its distance to one end of the DNA wasmeasured in order to assign a genomic position. In order to account forthe large variation in stretching factor between different DNA moleculesthe measured locations were normalized to units of percentage of thewhole genome (100% representing the far end of the phage genome at 48500base pairs). By dividing the distance of the tag location from the DNAend point by the total length of the template DNA molecule, a normalizedvalue for all detected tags is calculated, allowing statistical analysisof the pooled data as shown hereinbelow.

Experimental Results

β-GT was used to tag 5hmC sites with a fluorescent reporter molecule(labeling agent). The enzyme was fed with a synthetic cofactorUDP-6-N₃-Glu, resulting in covalent attachment of a functional azide atthe 5hmC site (FIG. 2).

This azide was further reacted with an ALEXA FLUOR® alkyne via a “click”chemistry reaction to generate the fluorescently labeled 5hmC (FIG. 2).The resulting DNA product had fluorescence and absorbance proportionalto the content of 5hmC residues.

To demonstrate the viability of such an approach 10 specific 5hmC siteswithin the 48.5-kb genome of lambda bacteriophage were engineered.Hydroxymethylated cytosine nucleotides were incorporated by a DNApolymerase (Vent exo-) into nicks induced along the genome at GCTCTTC(SEQ ID NO: 5) sites by the endonuclease BspQI. The hydroxymethylatedsites along the DNA were labeled after glucosylation with ALEXA FLUOR®555. The DNA was stained with YoYo-1 intercalating dye and extended onmodified glass surfaces for imaging¹³. Dual-color fluorescence images ofthe sample revealed that the lambda phage DNA was decorated withfluorescent spots (FIG. 3B). In order to map the positions of the spotsalong the DNA and correlate them with the expected 5hmC pattern, onlymolecules that were more than 80 pixels long were analyzed. This lengthcorresponds to a minimum threshold of 70% extension relative to the 17μm, full length of the genome. At this extension the labels maintainedtheir relative positions, and labeling could be quantitatively assessed.The expected pattern and several examples of individual genomesdecorated with multiple fluorescent spots indicating 5hmC sites areshown in FIGS. 3A and 3B. The expected 5hmC pattern is clearlyreconstructed by the fluorescent labels, indicating that labeling washighly specific.

For mapping the 5hmC sites, fluorescent spots were localized by2D-Gaussian fitting and their positions relative to the DNA extremitieswere measured. To compensate for the poor extension uniformity of theDNA molecules, the positions relative to the full length of the genomewere represented as percentages from the whole. Out of the 157 moleculesthat passed the length threshold, 93 molecules with clearly visibleorientations that were labeled at four or more positions along thegenome were analyzed. A histogram of all detected fluorescent spots(N=512) shows clear resemblance to the reference map, and multi-peakGaussian fitting of the histogram is in good agreement with expectedpositions (FIGS. 3A and 3B).

5hmC was labeled in DNA extracted from mouse brain and kidney (FIGS. 3Cand 3D). Both tissues are reported to be relatively rich in 5hmC¹⁶ andfluorescent spots indicating individual hmC residues are clearly seenalong the genomic fragments.

To test the efficiency of the labeling procedure, a ratiometricmeasurement was performed, that compared the fluorescence signal fromPCR synthesized DNA containing labeled 5hmC residues to that ofidentical DNA in which 5hmC bases were substituted with an ALEXA FLUOR®647 fluorophore. The latter served as a control that represents 100%labeling efficiency. An ALEXA FLUOR® 488 pre-labeled PCR primer was usedfor the reaction in order to report on the total amount of DNA analyzed.ALEXA FLUOR® 647 was used for the 5hmC labeling. The two sets of DNAmolecules were run side by side on a 3% agarose gel, and the fluorescentDNA bands were imaged on a multicolor gel scanner. Band intensity in thegreen channel, representing the single ALEXA FLUOR® 488 present in allmolecules, allowed normalization for the total amount of DNA in thedetected bands. The degree of 5hmC labeling was deduced from thefluorescence intensities of the bands in the red channel, by calculatingthe ratio of ALEXA FLUOR® 647 fluorescence levels between the controland the 5hmC-labeled samples. Analysis of the relative intensity levelsindicated a total labeling efficiency of 84%.

Another useful feature of the present labeling scheme is the ability touse a simple UV-Vis spectrophotometer in order to quantify global 5hmClevels in DNA. In the absorption spectrum, both the labeling fluorophoreand the DNA bases themselves have a characteristic absorption band thatcan be used to directly quantify the amount of 5hmC relative to thetotal DNA content. One example is presented in FIG. 4A. The labeled DNAhas an absorbance spectrum featuring distinct maxima at 260 nm (for DNA)and 647 nm (for labeled 5hmC). This potentially provides a sensitiveassay for quantifying global 5hmC levels in genomic DNA.

Since 5hmC levels vary greatly between different tissues and differentcell lines, with typical values from around 0.01% for HeLa cells and upto 0.65% for hmC/dG human brain tissue¹⁷, it was verified that themethod is sensitive enough to access biologically relevant genomic 5hmCcontent. Four calibration samples with total nucleotide to 5hmC ratiosbetween 1:500 (0.2%) and 1:5000 (0.02%) were prepared and used in theprotocol to tag glucosilated 5hmC with ALEXA FLUOR® 647. Theconcentration of the calibration samples were on the order of 600 to3500 ng/μL of DNA. Only 1 μl of sample was used for each UV-Visabsorption measurement, requiring amounts of DNA easily obtained fromless than 1 mg of tissue by standard DNA extraction kits. After spectrawere taken for all samples, a calibration curve was built using theabsorption data (FIG. 4B).

FIG. 4B demonstrates a full linearity of the relative absorbance at 260nm and 647 nm of an ALEXA FLUOR® 647-labeled DNA and the amount of 5hmCin the DNA, indicating that relative absorbance can be used as asensitive measurement of quantifying 5hmC.

In order to verify that labeling is specific and that no residualabsorbance at 647 nm occurs due to non-specific binding, an identicalexperiment was conducted only substituting the UDP-glucose-azide with astandard UDP-glucose which is not reactive towards the alkyne modifiedALEXA FLUOR® 647 dye. No residual absorption was detected in the controlsample; indicating that fluorescent labeling of 5hmC residues mayprovide a rapid and facile mean to quantify total 5hmC content ingenomic DNA from various sources.

The measurement is performed on a standard spectrophotometer readilyavailable in most labs and requires small amounts of genetic material.As opposed to previously published techniques that require furthertimely post-processing such as pull-down, RT-PCR, HPLC or enzymaticsignal amplification such as ELISA¹⁸ the reported technique deliversrapid and unambiguous results and lends itself readily to automatedanalysis in a high-throughput multi-well format.

Example 2

5hmC Labeling is Sensitive and can be Done in High Throughput Settings

Example 1 above establishes the use of some embodiments of the presentmethodology for the quantification of global % hmC in a given sample.The technique is based on covalent labeling of hmC moieties by anenzymatic reaction, which is followed by the Huisgen cycloaddition of analkyne to an azide moiety. Fluorescently labeled alkynes are used tofluorescently label hmC so that a ratiometric measurement of theabsorption intensities of the fluorophore relative to the DNA, at 260nm, can be obtained. These measurements were conducted on a nanodropspectrophotometer. A drop of 1 μl containing 1650 ng/μl of DNA wasrequired in order to detect 0.02% hmC in a given sample. This is ahighly concentrated sample which could sometimes be challenging toachieve. Each sample is measured separately since only one drop could bemeasured at a time.

Following is an improved ratiometric detection method which is based onthe ratio between the fluorescence intensity (rather than theabsorption) of a hmC-labeled DNA sample and the absorption intensity ofthe DNA, at 260 nm (FIGS. 5A-5B). The value obtained is compared to aratiometric calibration curve, prepared in the same manner for sampleswith known % hmC (FIG. 6A, insert). The fluorescent measurements aremore sensitive than the absorption measurements of labeled hmC allowingto detect down to 0.004% hmC/dN from a DNA sample extracted from theliver, with a sample concentration of only 136 ng/ul in 20 μl volume(2.7 μg) and 0.02% hmC/dN from a DNA sample concentration of only 82ng/ul in 20 μl volume (1.6 μg) (FIG. 6B).

The present methodology was further assessed in high throughputsettings. Measurements were conducted on a 384 well multiplate using amultiplate reader Tecan infinit M200. This allows measuring multiplesamples in one scan, eliminating errors that may be extracted frominstrumental factors.

In addition to global hmC quantifications, it was shown above in Example1, that it is possible to optically image hmC sites in single DNAfragments, stretched on glass slides. The following experiments haveshown that single molecule experiments allows the detection of not onlyextremely low hmC amounts such as in human peripheral bloodmononucleated cell (PBMC) (FIG. 7A) and spleen (FIG. 7B) but alsoinhomogeneity in the amount or distribution of hmC in DNA fragmentsextracted from the same tissue sample: DNA extracted from the spleen andfrom PBMC (FIGS. 7A and 7B) had, in most fragments, very low frequencyof hmC labeling (orange arrows), whereas, few fragments had irregularlyhigh hmC labeling (blue arrows). In the brain, most DNA fragments hadhigh hmC labeling (FIG. 7C). This data, which points out for variationswithin populations could only be detected by single molecule techniques.

Finding regions of enriched hmC moieties on single DNA strands must beaccompanied by a system that would allow the identification of theposition of these regions within the entire genome, relative to specificgenes within different chromosomes. Such system may be based on thecreation of a specific DNA signature map of a typical pattern. Asignature map or a DNA barcoding may be achieved by sequence-specificnicking enzymes, which make nicks in DNA fragments in positions adjacentto their recognition sights. Further enzymatic nuclear polymerizationreaction allows the incorporation of fluorescently labeled nucleotidesin the region of the nicked positions, forming a sequence-specificbarcoding of DNA strands (FIGS. 8A-8B, green label). FIGS. 8A-8B showDNA strands, stretched on glass slides (Blue) from Zebra fish (A) andfrom mouse brain (B) that are labeled both for hmC (pink) and for theBsPQI nicking enzyme recognition sites (green), allowing optical mappingof hmC sites.

REFERENCES Other References are Provided in the Document

-   1. S. Kriaucionis and N. Heintz, Science (New York, N.Y.), 2009,    324, 929-30.-   2. M. Tahiliani, K. P. Koh, Y. Shen, W. A. Pastor, H. Bandukwala, Y.    Brudno, S. Agarwal, L. M. Iyer, D. R. Liu, L. Aravind, and A. Rao,    Science (New York, N.Y.), 2009, 324, 930-5.-   3. Y.-F. He, B.-Z. Li, Z. Li, P. Liu, Y. Wang, Q. Tang, J. Ding, Y.    Jia, Z. Chen, L. Li, Y. Sun, X. Li, Q. Dai, C.-X. Song, K. Zhang, C.    He, and G.-L. Xu, Science (New York, N.Y.), 2011, 333, 1303-7.-   4. C.-X. Song, K. E. Szulwach, Y. Fu, Q. Dai, C. Yi, X. Li, Y. Li,    C.-H. Chen, W. Zhang, X. Jian, J. Wang, L. Zhang, T. J. Looney, B.    Zhang, L. A. Godley, L. M. Hicks, B. T. Lahn, P. Jin, and C. He,    Nature biotechnology, 2011, 29, 68-72.-   5. M. Munzel, D. Globisch, and T. Carell, Angewandte Chemie    (International ed. in English), 2011, 50, 6460-8.-   6. B. Teague, M. S. Waterman, S. Goldstein, K. Potamousis, S.    Zhou, S. Reslewic, D. Sarkar, A. Valouev, C. Churas, J. M. Kidd, S.    Kohn, R. Runnheim, C. Lamers, D. Forrest, M. A. Newton, E. E.    Eichler, M. Kent-First, U. Surti, M. Livny, and D. C. Schwartz,    Proceedings of the National Academy of Sciences of the United States    of America, 2010, 107, 10848-53.-   7. M. Levy-Sakin and Y. Ebenstein, Current Opinion in Biotechnology,    2013, null.-   8. R. K. Neely, J. Deen, and J. Hofkens, Biopolymers, 2011, 95,    298-311.-   9. E. T. Lam, A. Hastie, C. Lin, D. Ehrlich, S. K. Das, M. D.    Austin, P. Deshpande, H. Cao, N. Nagarajan, M. Xiao, and P.-Y. Kwok,    Nature biotechnology, 2012, 30, 771-6.-   10. Y. Michaeli and Y. Ebenstein, Nature biotechnology, 2012, 30,    762-3.-   11. Y. Ebenstein, N. Gassman, S. Kim, J. Antelman, Y. Kim, S. Ho, R.    Samuel, X. Michalet, and S. Weiss, Nano Letters, 2009, 9, 1598-1603.-   12. S. Kim, A. Gottfried, R. R. Lin, T. Dertinger, A. S. Kim, S.    Chung, R. A. Colyer, E. Weinhold, S. Weiss, and Y. Ebenstein,    Angewandte Chemie (International ed. in English), 2012, 51, 3578-81.-   13. J. M. Sidorova, N. Li, D. C. Schwartz, A. Folch, and R. J.    Monnat, Nature protocols, 2009, 4, 849-61.-   14. A. R. Hastie, L. Dong, A. Smith, J. Finklestein, E. T. Lam, N.    Huo, H. Cao, P.-Y. Kwok, K. R. Deal, J. Dvorak, M.-C. Luo, Y. Gu,    and M. Xiao, PloS one, 2013, 8, e55864.-   15. C. E. Nestor, R. Ottaviano, J. Reddington, D. Sproul, D.    Reinhardt, D. Dunican, E. Katz, J. M. Dixon, D. J. Harrison,    and R. R. Meehan, Genome research, 2012, 22, 467-77.-   16. A. Szwagierczak, S. Bultmann, C. S. Schmidt, F. Spada, and H.    Leonhardt, Nucleic acids research, 2010, 38, e181.-   17. W. Li and M. Liu, Journal of nucleic acids, 2011, 2011, 870726.-   18. M. R. Branco, G. Ficz, and W. Reik, Nature reviews. Genetics,    2012, 13, 7-13.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting. In addition, any priority document(s) of this applicationis/are hereby incorporated herein by reference in its/their entirety.

What is claimed is:
 1. A method of preparing UDP-6-N₃-Glucose, themethod comprising subjecting 6-azidoglucose to an enzymatic catalysis byN-acetylhexoseamine 1-kinase (NahK) in the presence of ATP to therebyobtain a phosphorylated 6-azidoglucose; and subjecting thephosphorylated 6-azidoglucose to enzymatic catalysis byuridyltransferase (GlmU) in the presence of UTP.
 2. A method of labelingthe epigenetic modification 5-hydroxymethyl-cytosine along a DNAmolecule, the method comprising attaching to said DNA molecule a5-hydroxymethyl-cytosine specific labeling agent, wherein said attachingcomprises: incubating said DNA molecule with UDP-6-N₃-Glucose preparedaccording to the method of claim 1 and β-glucosyltransferase, to therebyobtain a 5-hydroxymethyl-cytosine glycosylated by a 6-azidoglucoseresidue in said DNA molecule; and reacting said DNA molecule comprisingsaid 5-hydroxymethyl-cytosine glycosylated by a 6-azidoglucose residuewith a labeling agent derivatized by a reactive group which ischemically compatible with the azide group of said 6-azidoglucoseresidue.
 3. The method of claim 2, wherein said labeling agentderivatized by said reactive group is a fluorescent labeling agent. 4.The method of claim 2, further comprising attaching to said DNA moleculean additional labeling agent distinct of said 5-hydroxymethyl-cytosinespecific labeling agent.
 5. The method of claim 4, wherein saidadditional labeling agent is a 5-methyl-cytosine specific labelingagent.
 6. The method of claim 4, wherein said additional labeling agentis an epigenetic modification specific labeling agent.
 7. The method ofclaim 4, wherein said additional labeling agent is a non-epigeneticmodification specific labeling agent.
 8. The method of claim 7, whereinsaid non-epigenetic modification specific labeling agent is afluorescent agent which binds to nucleic acids.
 9. The method of claim2, not comprising subjecting said DNA molecule to fragmentation.
 10. Themethod of claim 2, wherein said reactive group is an alkyne.
 11. Themethod of claim 10, wherein attaching said labeling agent to said DNAmolecule is effected by a click chemistry.
 12. The method of claim 2,wherein said reacting is free of a copper catalyst.
 13. The method ofclaim 2, wherein said DNA molecule is a genomic DNA molecule.
 14. Themethod of claim 13, wherein said DNA molecule is longer than 20 Kb. 15.The method of claim 13, wherein said DNA molecule is longer than 40 Kb.16. The method of claim 2, further comprising detecting the5-hydroxymethyl-cytosine specific labeling agent.
 17. The method ofclaim 16, wherein said detecting is effected in a high throughputsetting of at least 300 DNA samples.
 18. A method of in-situ imaging aDNA molecule, the method comprising: (a) attaching a5-hydroxymethyl-cytosine specific labeling agent to said DNA moleculeaccording to the method of claim 2; and (b) subjecting said DNA moleculeto an imaging method suitable for detecting said5-hydroxymethyl-cytosine specific labeling agent.
 19. The method ofclaim 18, wherein said labeling agent is a fluorescent labeling agentsaid imaging method is a fluorescent imaging.
 20. The method of claim18, further comprising generating an optical image of said DNA moleculefollowing said imaging.